Project Summary Double-hit lymphoma (DHL) and double-expressing lymphoma (DEL) are aggressive subtypes of diffuse large B cell lymphoma (DLBCL) that are characterized by translocations or amplifications of both MYC and BCL2 oncogenes, or that co-overexpress MYC and BCL2 oncoproteins, respectively. MYC and BCL2 drive cancer cell growth and metabolism, or confer a marked resistance to apoptosis, respectively. Accordingly, patients with DHL or DEL respond poorly to chemotherapy and targeted therapies, and currently lack effective treatment options; indeed DHL and DEL are currently considered incurable. Thus, there is an urgent need to define new therapeutically tractable vulnerabilities for the treatment of DHL and DEL. Recently, we modeled DHL using a platform that includes bone marrow stroma and that mimics the DHL tumor microenvironment. Using this platform we implemented unbiased activity-based proteomic profiling, drug screens, RNA-seq and ChIP-seq studies to identify essential pathways and targets that are manifest in DHL. Quite strikingly, these analyses revealed that DHL have a unique super-enhancer (SE) landscape, where SE manifest in DHL are associated with genes that control the lymphoma cell fate or oncogenic signaling. Further, these screens revealed that DHL and DEL cell lines and primary patient specimens are highly sensitive to inhibitors of the general transcription apparatus. In particular, all DHL and DEL cells are exquisitely sensitive to THZ1, a newly identified covalent inhibitor of cyclin- dependent kinase 7 (CDK7) that functions as a transcriptional co-factor and that phosphorylates the C-terminal domain of RNA polymerase-II. Furthermore, our studies with a selective inhibitor of CDK9 coined NVP2 revealed that DHL and DEL cells survival also requires the activity of this kinase, which regulates transcriptional elongation. Notably our new findings have established that both CDK7 and CDK90 function are essential to maintain MYC expression in models of DHL and DEL. We hypothesize that MYC-, CDK7- and CDK9- dependent transcription and BCL-2 overexpression cooperatively drive the aggressive phenotypes of DHL and DEL and, accordingly, that combined inhibition of CDK7 or CDK9 and BCL-2 triggers synthetic lethality in these aggressive lymphomas. Using our cell-based platform, DHL and DEL cell line models, DHL patient-derived xenografts (PDX) and a syngeneic mouse DHL model that are available in our laboratories, our respective expertise, and our unique access to large numbers of primary DHL patient specimens, we will address the role of CDK7 and CDK9 in the maintenance of DHL, and we will define the mechanism by which CDK7/CDK9 sustains the expression of oncogenic drivers in this lethal malignancy. Finally, we will strategically target the transcriptional machinery and pre-clinically validate a therapeutic strategy that combines drugs that disable CDK7, CDK9 and BCL2 as a synthetic therapeutic approach to treat DHL and DEL. !